Across the diverse family of flavoenzymes, the isoalloxazine cofactor was found to display extremely diverse redox properties, both with respect to the absolute value of the potential regime wherein it operates and to its redox cooperativity, that is, the relative positioning of its individual 1-electron transitions. Taking together electrochemical data and 3D structural information reported for selected representatives of the flavoenzyme family, we assessed the contribution of pK value modifications at the three protonatable nitrogens of the isoalloxazine moiety. While the absolute value of the redox regime appears only weakly dependent on such pK modifications, the diversity of redox cooperativity is readily rationalized by (protein-induced) stabilization/destabilization of the proton primarily on N5 and to lesser degrees on N1 and N3. The mathematical formalism underlying the interdependence of pK values and redox midpoint potentials is subsequently extended to representatives of the family featuring extremely positive redox cooperativity (i.e., the electron bi/confurcating flavoenzymes). Observed structural idiosyncrasies of these cases were found to rationalize the extremely strong inversion (ΔE ≪ −800 mV) of 1-electron midpoint potentials in the framework of this formalism.

Anaerobic ammonium oxidizing bacteria make a living oxidizing ammonium with nitrite as electron acceptor, intermediates nitric oxide and hydrazine, and end product dinitrogen gas. Hydrazine is a biologically unique free intermediate in this metabolism, and is produced by the enzyme hydrazine synthase. Crystallization of ‘ Candidatus Kuenenia stuttgartiensis’ hydrazine synthase allowed for an initial hypothesis of its reaction mechanism. In this hypothesis, nitric oxide is first reduced to hydroxylamine after which hydroxylamine is condensed with ammonium to form hydrazine. Hydrazine synthase is a tetraheme cytochrome c , containing two proposed active site hemes (γI & αI) in the γ- and α-subunit, respectively, connected by an intra-enzymatic tunnel. Here we combined the data from electrochemistry-induced Fourier transform infrared (FTIR) spectroscopy, EPR and optical spectroscopy to shed light on the redox properties and protein dynamics of hydrazine synthase in the context of its reaction mechanism. Redox titrations revealed two low potential low spin hemes with midpoint potentials of ∼-360 mV and ∼-310 mV for heme αII and γII, respectively. Heme γI showed redox transitions in the range of 0 mV, consisting of both low spin and high spin characteristics in optical and EPR spectroscopy. Electrochemistry-induced FTIR spectroscopy indicated an aspartic acid ligating a OH - /H 2 O at the heme γI axial site as a possible candidate for involvement in this mixed spin characteristic. Furthermore, EPR spectroscopy confirmed the ability of heme γI to bind NO in the reduced state. Heme αI exhibited a rhombic high spin signal, in line with its ligation by a proximal tyrosine observed in the crystal structure. Redox titrations down to −610 mV nor addition of dithionite resulted in the reduction of heme αI, indicating a very low midpoint potential for this heme. In vivo chemistry at this heme αI, the candidate for the comproportionation of hydroxylamine and ammonium, is thus likely to be initiated solely on the oxidized heme, in contrast to previously reported DFT calculations. The reduction potentials of the γ-subunit hemes were in line with the proposed electron transfer of heme γII to heme γI for the reduction of NO to hydroxylamine (E 0 ’ = − 30 mV).

The dominant organisms in modern oxic ecosystems rely on respiratory quinones with high redox potential (HPQs) for electron transport in aerobic respiration and photosynthesis. The diversification of quinones, from low redox potential (LPQ) in anaerobes to HPQs in aerobes, is assumed to have followed Earth's surface oxygenation ~2.3 billion years ago. However, the evolutionary origins of HPQs remain unresolved. Here, we characterize the structure and biosynthetic pathway of an ancestral HPQ, methyl-plastoquinone (mPQ), that is unique to bacteria of the phylum Nitrospirota. mPQ is structurally related to the two previously known HPQs, plastoquinone from Cyanobacteriota/chloroplasts and ubiquinone from Pseudomonadota/mitochondria, respectively. We demonstrate a common origin of the three HPQ biosynthetic pathways that predates the emergence of Nitrospirota, Cyanobacteriota, and Pseudomonadota. An ancestral HPQ biosynthetic pathway evolved ≥ 3.4 billion years ago in an extinct lineage and was laterally transferred to these three phyla ~2.5 to 3.2 billion years ago. We show that Cyanobacteriota and Pseudomonadota were ancestrally aerobic and thus propose that aerobic metabolism using HPQs significantly predates Earth's surface oxygenation. Two of the three HPQ pathways were later obtained by eukaryotes through endosymbiosis forming chloroplasts and mitochondria, enabling their rise to dominance in modern oxic ecosystems.

The heterotrimeric [FeFe]‑hydrogenase (TmHydABC) from Thermotoga maritima synergistically uses electrons from ferredoxin and NADH to reduce protons to hydrogen enabling the anaerobic metabolism of carbohydrates and other molecules. The precise mechanism by which this flavin-based electron confurcation is achieved is still unknown. Here, the redox properties of the cofactors in TmHydABC were characterized via chemical and electrochemical redox titrations monitored by EPR- and UV/Vis-spectroscopy. Deconvolution of nine out of eleven iron‑sulfur clusters harboured by the apo-enzyme was achieved by (a) exploiting the distinct relaxation properties (and hence temperature-dependencies) of the EPR-signals, (b) making use of spectral idiosyncrasies of [4Fe-4S] versus thioredoxin-like [2Fe2S] clusters in UV/Vis-spectroscopy and (c) using the individual HydA, HydB and HydC subunits and C-terminal domains of HydA and HydB. Furthermore, electrostatic interactions between certain neighbouring clusters and a paramagnetic interaction between the flavin and one of the iron‑sulfur clusters were revealed. The electrochemical parameters of the flavin were obtained both via UV/Vis-spectroscopy and EPR-analysis of its semi-reduced state in isolated HydB. This semi-reduced state was found to correspond to a neutral flavosemiquinone and a pKa (attributed to the N5-proton) was extracted from the pH-dependence of its redox midpoint potential. The electrochemical properties of the flavin in the entire enzyme appear to differ significantly from those in the isolated HydB subunit. The flavin and iron‑sulfur cluster redox properties indicate a dynamic electrochemical landscape, potentially contributing to its mechanism.

The second law of thermodynamics leaves no doubt that life on planet Earth and its inherent substantial decrease in entropy is fundamentally based on mechanisms converting environmental free energy into the spatial and temporal order of metabolic processes. This argument holds for present life as much as it does for its very beginnings some 4 billion years ago. In this contribution, we try to strip down free energy conversion in extant life (known as "bioenergetics" to the biologists) to its basic principles with the aim to potentially retrodict the nature of the pre-biotic precursor which drove life into existence. We demonstrate that these basic principles are deeply rooted in aqueous electrochemistry and strongly rely on inorganic redox compounds. The question of life's emergence, generally considered to fall into the realm of organic chemistry, should therefore rather be recognized as an electrochemical problem and its ultimate elucidation will need to strongly implicate the community of electrochemical scientists.

Spin probe electron paramagnetic resonance spectroscopy is applied to characterize the dynamics of concentric hydration and mesophase solvent domains that surround proteins within the ice boundary in frozen aqueous solutions. The solvent dynamics are tuned by variation of temperature (190–265 K) and by the degree of ice boundary confinement, which is modulated by the volume of added cryosolvent (0–~50 Å separation distance from protein surface). Goals are to: (1) characterize the protein-coupled solvent dynamics on correlation time scales of ~ 10− 10 < τ < 10− 7 s, and spatial scales from protein surface to periphery of the surrounding solution, from the perspective of a free, small-molecule (~ 7 Å diameter) probe, and (2) reveal properties of the solvent-protein coupling that can be correlated with protein functions, that are measureable under the same conditions. Rotational mobility of the nitroxide spin probe, TEMPOL, resolves and tracks two solvent components, the protein-associated domain (PAD; akin to hydration layer) and surrounding mesodomain, through their distinct temperature- and confinement-dependent values of τ and normalized weight. Detailed protocols are described for simulation of two-component nitroxide EPR spectra, which are categorized by line shape regime and guided by a library of template spectra and simulation parameters derived from two model soluble globular proteins. The order-disorder transition in the PAD, which is a universal feature of protein-coupled solvent dynamics, provides a well-defined, tunable property for elucidating mechanism in solvent-protein-function dynamical coupling. The low-temperature mesodomain system and EPR spin probe method are generally applicable to reveal solvent contributions to a broad range of macromolecule-mediated biological processes.

In methanogenic archaea, the archetypical complex of heterodisulfide reductase (HdrABC) and hydrogenase (MvhAGD) couples the endergonic reduction of CO2 by H2 to the exergonic reduction of the CoB-S-S-CoM heterodisulfide by H2 via flavin-based electron bifurcation. Presently known enzymes containing HdrA(BC)-like components play key roles in methanogenesis, acetogenesis, respiratory sulfate reduction, lithotrophic reduced sulfur compound oxidation, aromatic compound degradation, fermentations, and probably many further processes. This functional diversity is achieved by a modular architecture of HdrA(BC) enzymes, where a big variety of electron input/output modules may be connected either directly or via adaptor modules to the HdrA(BC) components. Many, but not all HdrA(BC) complexes are proposed to catalyse a flavin-based electron bifurcation/confurcation. Despite the availability of HdrA(BC) crystal structures, fundamental questions of electron transfer and energy coupling processes remain. Here, we address the common properties and functional diversity of HdrA(BC) core modules integrated into electron-transfer machineries of outstanding complexity.

Metal-bearing minerals are an integral part of almost all "metabolism-first"-type scenarios for the emergence of life which consider that life is better defined by what it does than what it is made from. Since metals are formidable catalysts, these scenarios stipulate that early metabolic reactions (and prominently the reduction of CO2 to yield biomass) were performed by (mainly transition) metals contained in certain minerals. Metabolismfirst scenarios stand in opposition to primordial soup hypotheses which envisage prebiotic synthesis of organic molecules as building blocks for life to be the salient feature enabling life to come into being. A critical analysis of the historical roots of these emergence of life hypotheses highlights fundamental inconsistencies prompting us to appeal to basic thermodynamic principles to provide rigorous guidelines for developing contradiction-free models. Combining these guidelines with our present-day understanding of biological energy conversion, arguably the process most fundamental to all life, strongly suggests an expansion of previous mineral-based scenarios to include processes converting environmental redox tensions into phosphate-group-transfer disequilibria, i.e., the quintessential free energy converting mechanism of extant life. Based on their reported physicochemical and electrochemical properties, iron- (together with other transition metal-) based layered double oxyhydroxide (Fe-LDH) minerals such as fougerite are promising candidates to afford the required capacities and therefore may render previous mineral-based scenarios compliant with thermodynamic strictures.

The concomitant presence of two distinctive polypeptide modules, which we have chosen to denominate as the “Y-junction” and the “flavin” module, is observed in 3D structures of enzymes as functionally diverse as complex I, NAD(P)-dependent [NiFe]-hydrogenases and NAD(P)-dependent formate dehydrogenases. Amino acid sequence conservation furthermore suggests that both modules are also part of NAD(P)-dependent [FeFe]-hydrogenases for which no 3D structure model is available yet. The flavin module harbours the site of interaction with the substrate NAD(P) which exchanges two electrons with a strictly conserved flavin moiety. The Y-junction module typically contains four iron-sulphur centres arranged to form a Y-shaped electron transfer conduit and mediates electron transfer between the flavin module and the catalytic units of the respective enzymes. The Y-junction module represents an electron transfer hub with three potential electron entry/exit sites. The pattern of specific redox centres present both in the Y-junction and the flavin module is correlated to present knowledge of these enzymes' functional properties. We have searched publicly accessible genomes for gene clusters containing both the Y-junction and the flavin module to assemble a comprehensive picture of the diversity of enzymes harbouring this dyad of modules and to reconstruct their phylogenetic relationships. These analyses indicate the presence of the dyad already in the last universal common ancestor and the emergence of complex I's EFG-module out of a subgroup of NAD(P)- dependent formate dehydrogenases.

Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally-occurring contracted Cys-Lys-Cys-His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.

SummaryDuring daffodil flower development, chloroplasts differentiate into photosynthetically inactive chromoplasts having lost functional photosynthetic reaction centers. Chromoplasts exhibit a respiratory activity reducing oxygen to water and generating ATP. Immunoblots revealed the presence of the plastid terminal oxidase (PTOX), the NAD(P)H dehydrogenase (NDH) complex, the cytochrome b6f complex, ATP synthase and several isoforms of ferredoxin‐NADP+ oxidoreductase (FNR), and ferredoxin (Fd). Fluorescence spectroscopy allowed the detection of chlorophyll a in the cytochrome b6f complex. Here we characterize the electron transport pathway of chromorespiration by using specific inhibitors for the NDH complex, the cytochrome b6f complex, FNR and redox‐inactive Fd in which the iron was replaced by gallium. Our data suggest an electron flow via two separate pathways, both reducing plastoquinone (PQ) and using PTOX as oxidase. The first oxidizes NADPH via FNR, Fd and cytochrome bh of the cytochrome b6f complex, and does not result in the pumping of protons across the membrane. In the second, electron transport takes place via the NDH complex using both NADH and NADPH as electron donor. FNR and Fd are not involved in this pathway. The NDH complex is responsible for the generation of the proton gradient. We propose a model for chromorespiration that may also be relevant for the understanding of chlororespiration and for the characterization of the electron input from Fd to the cytochrome b6f complex during cyclic electron transport in chloroplasts.

We here review the extraordinary mineralogical properties of green rusts and their naturally occurring form, fougerite, and discuss the pertinence of these properties within the alkaline hydrothermal vent (AHV) hypothesis for life's emergence. We put forward an extended version of the AHV scenario which enhances the conformity between extant life and its earliest progenitor by extensively making use of fougerite's mechanistic and catalytic particularities.

Electron bifurcation is here described as a special case of the continuum of electron transfer reactions accessible to two-electron redox compounds with redox cooperativity. We argue that electron bifurcation is foremost an electrochemical phenomenon based on (a) strongly inverted redox potentials of the individual redox transitions, (b) a high endergonicity of the first redox transition and (c) an escapement-type mechanism rendering completion of the first electron transfer contingent on occurrence of the second one. This mechanism is proposed to govern both the traditional quinone-based and the newly discovered flavin-based versions of electron bifurcation. Conserved and variable aspects of the spatial arrangement of electron transfer partners in flavoenzymes are assayed by comparing the presently available 3D structures. A wide sample of flavoenzymes is analyzed with respect to conserved structural modules and three major structural groups are identified which serve as basic frames for the evolutionary construction of a plethora of flavin-containing redox enzymes. We argue that flavin-based and other types of electron bifurcation are of primordial importance to free energy conversion, the quintessential foundation of life, and discuss a plausible evolutionary ancestry of the mechanism.

The most abundant transition metal in biological systems is iron. It is incorporated into protein cofactors and serves either catalytic, redox or regulatory purposes. Anaerobic ammonium oxidizing (anammox) bacteria rely heavily on iron-containing proteins - especially cytochromes - for their energy conservation, which occurs within a unique organelle, the anammoxosome. Both their anaerobic lifestyle and the presence of an additional cellular compartment challenge our understanding of iron processing. Here, we combine existing concepts of iron uptake, utilization and metabolism, and cellular fate with genomic and still limited biochemical and physiological data on anammox bacteria to propose pathways these bacteria may employ.

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

The electrochemical parameters of all cofactors in the supercomplex formed by the Rieske/cytb complex and the SoxM/A-type O2-reductase from the menaquinone-containing Firmicute Geobacillus stearothermophilus were determined by spectroelectrochemistry and EPR redox titrations. All redox midpoint potentials (Em) were found to be lower than those of ubi- or plastoquinone-containing systems by a value comparable to the redox potential difference between the respective quinones. In particular, Em values of +200mV, −360mV, −220mV and −50mV (at pH7) were obtained for the Rieske cluster, heme bL, heme bH and heme ci, respectively. Comparable values of −330mV, −200mV and +120mV for hemes bL, bH and the Rieske cluster were determined for an anaerobic Firmicute, Heliobacterium modesticaldum. Thermodynamic constraints, optimization of proton motive force build-up and the necessity of ROS-avoidance imposed by the rise in atmospheric O2 2.5billionyears ago are discussed as putative evolutionary driving forces resulting in the observed redox upshift. The close conservation of the entire redox landscape between low and high potential systems suggests that operation of the Q-cycle requires the precise electrochemical tuning of enzyme cofactors to the quinone substrate as stipulated in P. Mitchell's hypothesis.

The eukaryotic Elongator complex participates in modification of uridines in tRNAs. Structural and functional work on a bacterial Elp3, the catalytic subunit of Elongator, provides insight into the function and mechanism of this important enzyme. During translation elongation, decoding is based on the recognition of codons by corresponding tRNA anticodon triplets. Molecular mechanisms that regulate global protein synthesis via specific base modifications in tRNA anticodons are receiving increasing attention. The conserved eukaryotic Elongator complex specifically modifies uridines located in the wobble base position of tRNAs. Mutations in Elongator subunits are associated with certain neurodegenerative diseases and cancer. Here we present the crystal structure of D. mccartyi Elp3 (DmcElp3) at 2.15-Å resolution. Our results reveal an unexpected arrangement of Elp3 lysine acetyltransferase (KAT) and radical S-adenosyl methionine (SAM) domains, which share a large interface and form a composite active site and tRNA-binding pocket, with an iron–sulfur cluster located in the dimerization interface of two DmcElp3 molecules. Structure-guided mutagenesis studies of yeast Elp3 confirmed the relevance of our findings for eukaryotic Elp3s and should aid in understanding the cellular functions and pathophysiological roles of Elongator.

The cyanobacterium Synechocystis PCC 6803 possesses three Rieske isoforms: PetC1, PetC2 and PetC3. While PetC1 and PetC2 have been identified as alternative subunits of the cytochrome b(6)f complex (ben, PetC3 was localized exclusively within the plasma membrane. The spatial separation of PetC3 from the photosynthetic and respiratory protein complexes raises doubt in its involvement in bioenergetic electron transfer.Here we report a detailed structural and functional characterization of the cyanobacterial PetC3 protein family indicating that PetC3 is not a component of the b(6)f and the photosynthetic electron transport as implied by gene annotation. Instead PetC3 has a distinct function in cell envelope homeostasis. Especially proteomic analysis shows that deletion of petC3 in Synechocystis PCC 6803 primarily affects cell envelope proteins including many nutrient transport systems. Therefore, the observed downregulation in the photosynthetic electron transport mainly caused by photosystem 2 inactivation-might constitute a stress adaptation. Comprehensive in silico sequence analyses revealed that PetC3 proteins are periplasmic lipoproteins tethered to the plasma membrane with a subclass consisting of soluble periplasmic proteins, i.e. their N-terminal domain is inconsistent with their integration into the b(6)f . For the first time, the structure of PetC3 was determined by X-ray crystallography at an atomic resolution revealing significant high similarities to non-b(6)f Rieske subunits in contrast to PetC1. These results suggest that PetC3 affects processes in the periplasmic compartment that only indirectly influence photosynthetic electron transport. For this reason, we suggest to rename "Photosynthetic electron transport Chain 3" (PetC3) proteins as "periplasmic Rieske proteins" (Prp). (C) 2016 Elsevier B.V. All rights reserved.